【作者】 迟强；

【导师】 梅兴国；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药剂学， 2008， 博士

【摘要】 随着生物技术的发展,蛋白多肽药物在现代疾病的预防和治疗中的应用日益广泛。蛋白多肽类药物稳定性差,口服或其它非注射给药途径生物利用度低,目前大部分蛋白多肽类药物采用注射给药;另一方面,由于该类药物在血液中的半衰期很短,注射后很快被清除或降解,因此需要频繁给药,患者顺应性较差。将蛋白多肽类药物制备成缓释注射剂不仅可以减少给药次数,提高患者依从性和治疗效果,同时还可以降低药物的毒副作用,减少总给药剂量,从而减轻病人的经济负担。胸腺五肽是一种临床上广泛应用的双向免疫调节剂,能够选择性诱导和促进T细胞的分化、增殖和成熟,调节T细胞亚群的比例,使CD4~+/CD8~+趋于正常。目前上市的胸腺五肽制剂以冻干粉针剂为主。但有研究证实,胸腺五肽的效能可以通过缓慢输注得到增强,因此开发能够持续释放药物的缓释制剂对于药物效能的发挥也是有利的。制备缓释制剂主要依据两个原理,一是减少溶出(Noyes-Whitney方程),一是减少扩散(Fick第一扩散定律)。根据Noyes-Whitney方程,减少药物的溶出则可以使溶出速度减慢;而降低药物的溶解度,主要基于两种途径,一种是制成不溶性盐和不溶性复合物,另一种是对药物进行结构修饰。本课题两种途径并行,一方面尝试将胸腺五肽直接制成不溶性复合物,另一方面则对胸腺五肽进行了成酯修饰。制备不溶性盐(酯)和不溶性复合物,制备方法比较简单,产品易于实现工业化生产,是一种广泛应用于上市产品的方法。因此,只要可以制备适宜的水不溶性盐(酯)而且其药效确切,未来的药物制剂仍会利用这种相对简单的方法继续生产缓释药物产品。本文首先尝试制备了胸腺五肽与羧甲基纤维素钠的水不溶性复合物。由于胸腺五肽水溶性大,不溶性复合物的生成量较少,收率较低,同时这种不溶性复合物的释放受释放介质离子强度的影响较大,在模拟组织液(Hank’s平衡盐溶液)中释放时间短,仅约6h,不能达到实验目的。为降低胸腺五肽的水溶性和提高其药效,本文采用化学方法对其进行了成酯修饰,得到不同链长的胸腺五肽酯化物。对各种胸腺五肽酯化产物进行了初步药效学研究。结果表明,酯化物的药效比原药有所增强。进而对酯化物的安全性和稳定性进行了考察,从中选择胸腺五肽乙酯作为模型药物,进一步制备不溶性复合物,最终制备出胸腺五肽乙酯的鞣酸盐复合物。这种复合物的油混悬液在体内外均显示出长效缓释作用。1胸腺五肽酯化物的合成、纯化与鉴别本文采用二氯亚砜法以不同链长的脂肪醇与胸腺五肽进行反应,对胸腺五肽末端羧基和侧链羧基同时进行酯化,得到了胸腺五肽的乙酯、丁酯、己酯和十二醇酯。采用重结晶、制备型薄层色谱或中压反相层析等方法对胸腺五肽酯化物进行了分离纯化,采用HPLC-ESI-MS法,辅以核磁共振和紫外光谱法对酯化产物进行了结构确证。2胸腺五肽酯化物的理化性质及稳定性研究胸腺五肽酯化物的理化性质与原药相比,发生较大的改变,脂溶性增大,水溶性明显降低,并且以胸腺五肽乙酯进行的血浆稳定性考察结果显示,其血浆半衰期稍有延长。酯化物的稳定性影响因素研究结果显示,酯化物对光较为敏感,高温、高湿环境对酯化物的稳定性也有一定影响。因此,酯化物应在低温避光干燥处存放。3胸腺五肽酯化物的药效学研究以MTT法考察了胸腺五肽酯化物对小鼠脾淋巴细胞增殖的影响,结果表明,胸腺五肽酯化物能够促进小鼠脾淋巴细胞的增殖,其作用强于胸腺五肽;并且酯化物的活性随脂肪链的延长而增强。另外,在氢化可的松所致大鼠免疫抑制模型上,以血浆SOD酶活力为指标,考察了胸腺五肽乙酯对免疫抑制大鼠SOD酶活力的影响。结果,胸腺五肽乙酯能够明显增强氢化可的松所致免疫抑制大鼠的血浆SOD酶活力,作用也强于胸腺五肽。4胸腺五肽酯化物的初步安全性评价采用小鼠为受试动物,以静脉注射和皮下注射两种给药途径对胸腺五肽乙酯和己酯的急性毒性进行了初步考察。结果表明,酯化物的毒性较胸腺五肽明显增大,静脉给药途径尤其明显,且己酯的毒性大于乙酯。但毒性反应持续时间较短,除急性死亡者外,一般在20 min内都会恢复正常。存活的小鼠在两周的观察期内均没有出现死亡,体重比给药前有所增加。皮下注射给药时胸腺五肽乙酯剂量达170 mg/kg(比静脉大一倍时),仍未见明显毒性反应,因而认为胸腺五肽乙酯皮下注射给药较为安全。在综合考虑药效、毒性和制备工艺的难易程度的基础上,确定胸腺五肽乙酯用于下一步的制剂研究。5胸腺五肽乙酯鞣酸盐的制备与体内外评价胸腺五肽乙酯与鞣酸可以形成稳定的不溶性复合物,该复合物在Hank’s平衡盐溶液中维持了较长的释放时间。大鼠体内药效学实验结果显示,皮下给予胸腺五肽乙酯鞣酸盐油溶液后血浆SOD活力明显增高,并持续约一周,SOD水平与普通制剂有显著性差异。本课题的创新之处在于,将胸腺五肽进行成酯修饰,得到药效增强,脂溶性增加的酯化产物,这些酯化产物目前还未见报道。另外,本课题在制备缓释制剂过程中,将药剂学方法和化学方法相结合,在通过化学修饰方法来实现缓控释方面进行了有益的探索。更多还原

【Abstract】 More and more peptides and proteins become main and important drugs along with the progress of genomics and protemics. However, compared with low molecular weight drugs, the distinct properties of them, such as easy degradation (e.g. by deamidation, oxidation and hydrosis), instability, low bioavailability and short half lives, make non-parenteral dosage forms not proper to deliver them. So it’s a significant task to deliver proteins and peptides to target organs and tissues. Nowadays the biopharmaceutical drugs on clinical use are supplied as sterile freeze-dried powder or injectable solutions. These formulations mentioned above are administered frequently and this results in the patients’poor compliance and high-cost therapy. To improve the patients’compliance and the therapy effect and decrease the administration frequency, developing sustained release formulations for these peptides and proteins is a choice of wisdom. The other advantages include: (1) maintaining minimum effective concentration and avoiding the peak-valley phenomenon which appeared regularly in the common formulations; (2) decreasing total dose and alleviating some toxic and adverse actions; (3) decreasing the patients’costs. Therefore, injectable sustained-release preparations have been studied extensively in the last two decades in the pharmaceutics field.Thymopentin (TP5), the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr, corresponds to residues 32-36 of the thymic hormone thymopoietin. It has pleiotropic actions including selective induction of early T-cell differentiation and regulation of neuromuscular transmission. Furthermore, TP5 has been shown to have immunoregulatory actions on peripheral T cells. The product available was mainly sterile freeze-dry powder. TP5 is freely soluble in water and quickly degraded by protease and aminopeptidase in plasma. TP5 potency in vivo was enhanced by slow infusion. This suggested that the peptide was suitable for development of a sustained release formulation.The methods frequently used in the preparation of depot injection include: (1) converting a drug into insoluble salt (or ester) or insoluble complex. (2) adding macromolecular polymer such as hyaluronic acid into the injection to increase the injection’s viscosity and delay the drug’s spread; (3) encapsulating peptides in liposomes and obtain a sustained release formulation of peptide; (4) incorporating drugs into solid particles to obtain a extended drug release. (5) in-situ gelling system.Among these methods mentioned above, the insoluble complex is relatively simple form. The method has been widely used in the product granted market clearance. As long as the insoluble salt (or ester) could be prepared and showed certain effect, this relatively simple method will be continued to produce sustained release products in the future.At first, we tried preparing a water insoluble complex combined with thymopentin and carboxymethylcellulose sodium. But the yield was low. We only obtained less amount of product due to the high solubility of thymopentin. Meanwhile, the vitro release of the insoluble complex was affected by the release medium obviously. The peptide released rapidly in simulated tissue fluid (Hank’s balanced salt solution) and almost completed in 6h. So we fail to prepare the insoluble complex directly combined with thymopentin and carboxymethylcellulose sodium. We have to look for other path to prepare sustained release formulation for thymopentin.To reduce water solubility of TP5, a chemical modification has been done. In our research, carboxyl groups in C terminal of thymopentin and side chain of aspartate are esterified. We synthesized a series of thymopentin ester derivates (ethyl, butyl, hexyl and lauryl ester). The preliminary pharmacodynamics and safety evaluated were carried out for the ester derivates. The stability of the ester derivates was investigated too. The ethyl ester was chosen for further pharmaceutics study, and at last we prepared an insoluble thymopentin ethyl ester-tannate salt complex. It shows characteristics of sustained release in vitro. So far some investigations have been made to increase the peptide’s stability and efficacy, including linear peptide analogues and derivates modified by polyethylene glycol. To our knowledge, no ester derivates of thymopentin are reported.1 Synthesis, purification and identification of thymopentin ester derivates In this dissertation, the synthesis of thymopentin ester derivates was carried out by thionyl chloride method. Thionyl chloride was first dissolved in absolute ethanol. The mixture was stirred at 0°C for some time. Thymopentin was added and continued stirring at 0°C. Allowed temperature to come back at room temperature spontaneously and stirred the reaction until the TP-5 disappeared. A thin-layer chromatography (TLC) was used to detect whether the reaction completed. Ninhydrin TLC reagent spray was employed to visualize the peptides on the TLC plate. The ethyl ester reaction mixture was concentrated in vacuo to a solid. The remaining n-butanol and n-hexanol in butyl ester and hexyl ester reaction mixture were removed under vacuum by forming azeotropic mixture with water. The crude product was purified using Flash column chromatography system. Acetonitrile and TFA were removed under vacuum, followed by lyophilization. The dry product was analyzed once again. The ester derivates were found to be >98% pure and were further characterized using LC-ESI-MS. Moreover, the purified ester derivates were used for in vitro and in vivo assay.2 The study on physical properties and stability of thymopentin ester derivates Esterification modification changes physical properties of thymopentin greatly.That is to say, the liposolubility of ester derivates increased and water solubility decreased obviously. The half life of TP5 in plasma was 1.3 min and TP-ET 2.0 min. The half life of ethyl ester was almost equivalent to that of TP5. The preliminary stability test showed that the ester derivates was sensitive to light, and temperature and moisture affected the stability of ester derivates to some extent. So the ester derivates should store in cool and dark places.3 Pharmacodynamics of thymopentin ester derivatesThe in vitro immunoregulatory activity of ester derivatives was assessed by mice lymphocyte proliferation test using MTT colorimetric assay. Lymphocytes of mice spleen suspended in RPMI 1640 medium with 10% heat inactivated fetal calf serum were set up in 96-wells cell culture cluster. Under the tested concentrations, optical densities of all test wells were higher than those of blank wells. Optical densities of all wells containing thymopentin ester derivates were higher than those containing TP5. These results indicated that ester derivates showed more potent lymphocyte proliferation activity compared with TP5. Moreover, the activities of ester derivates enhanced as the alkyl chains extended.Thymopentin ethyl ester was further used in plasma superoxide dismutase (SOD) activity assay. Hydrocortisone injection was administered subcutaneously to each rat at a dose of 50 mg/kg once daily for 7 consecutive days for construction of immunosuppression rats. All the rats were fasted for 8 h before the experiments, but had free access to water. From the eighth day, the rats were given different treatment for 14 days. 6 h after the last administration, a volume of 100μl blood sample was taken from the orbit venous plexus into heparinized tubes and immediately centrifuged at 4000 revolutions per minute for 10 min. The plasma was collected and stored at -20°C until assays. For each assay, a volume of 15μl plasma sample was used. All the processing and assay validation met the requirements of the kit instruction. The results showed that the SOD values of the immunosuppressive rats were significantly lowered as compared with those of the normal control rats, indicating the immunosuppressive model was stably established. The SOD values of the immunosuppressive rats after subcutaneous administration of TP5 ethyl ester were significantly increased as compared with those of the model control group. SOD values of TP5 control group were higher than those of model group (P<0.05). But SOD values of TP5 group were lower than those of TP5 ethyl ester group with medium doses and high doses (P<0.05), equivalent to those of TP-ET group with low doses (P>0.05).4 Preliminary safety of thymopentin ester derivatesMice in test groups were injected intravenously and subcutaneously with ethyl ester (TP-ET) and hexyl ester (TP-HEX) solution. A control group received the solvent only. The solvent comprised 30% propylene glycol and 70% physiological saline solution. Drug effects were investigated by observation of body weight changes and survival rates over 15 days. Animals were observed until day 15, after which they were euthanized and subjected to a gross necropsy. The LD50 of TP-ET was >85 mg/kg. Tremors and twisting were the signs most frequently observed in the animals treated with TP-ET at 85mg/kg and 125 mg/kg. The mice were observed no obvious toxic reaction when given TP-ET 170mg/kg subcutaneously, so TP-ET was considered safe for subcutaneous injection.When the mice received a single dose of TP-HEX, Ocular proptoses and tremors usually developed and could be accompanied by tachypnea. In the most severe signs, animals lost muscle tone and righting reflex, followed by death. At the rest days of the 15-day observation period, no mice died and the survivors in all treatment groups gained weight. There were no treatment related gross necropsy findings for either scheduled euthanasia or early death animals in the study.5 Preparation and in vitro/in vivo evaluation of thymopentin ethyl ester tannate Thymopentin ethyl ester could form a stable insoluble complex with tannic acid. The complex released for a prolonged period in Hank’s balanced salt solution. The rats were administered the complex oil suspension subcutaneously. The preparation showed a prolonged action period in elevating plasma SOD activity and the action maintained for about a week in vivo.The creative points of this work consist in that thymopentin ester derivates show enhanced immunoregulatory activity and increased liposolubility. This can be regarded as a new finding in chemical modification of thymopentin. On the other hand, this work provides a new example of“chemical pharmaceutics”in pharmaceutical developments.更多还原